Replisome pausing in mutagenesis

E. coli cells containing a temperature-sensitivednaE mutation, in the α-subunit of holoenzyme DNA polymerase III, do not survive at the restrictive temperature. Such cells may survive in the presence of thepcbA1 mutation, an allele of thegyrB gene. Such survival is dependent on an active DNA polymerase I. Evidence indicates that DNA polymerase I interacts directly in the replisome (REP·A). Despite normal survival for cells using thepcbA replication pathway after some type of DNA damage, we have noted a failure of damage-induced mutagenesis. Here we present evidence supporting a model of replisome pausing in cells dependent upon thepcbA replication pathway. The model argues that the (REP·A) complex pauses longer at the site of the lesion, allowing excision repair to occur completely. In the normal replication pathway (REP·E) bypass of the lesion occurs, fixing the mutation.     

Larva and pupa of Cyphopisthes descarpentriesi Paulian (Coleoptera: Scarabaeoidea: Ceratocanthidae) and their phylogenetic implications

Abstract Pupae and mature larvae of the Australian ceratocanthid beetle, Cyphopisthes descarpentriesi Paulian 1977, are described and extensively illustrated. This is the sixth species of the family for which immature stages are known and the first from the Australian region. Unlike other ceratocanthid larvae described before, those of Cyphopisthes Gestro lack stridulatory teeth on the middle and hind legs and any trace of a frontoclypeal suture on the cranium. Reduced one-segmented labial palpi in Cyphopisthes are unique in Scarabaeoidea. Monophyly of the family is not corroborated by larval characters. Absence of spiracular closing apparatus in larvae is reported in the family for the first time. Like pupae of Ceratocanthus White and Germarostes Paulian, those of Cyphopisthes have thoracic projections, but their shape and location are different. Spiracles are found on abdominal segments 2−4 of pupa; that on segment 2 differs in colour and location from the others.     

The evolutionary transformation of phyllopodous to stenopodous limbs in the Branchiopoda (Crustacea)--is there a common mechanism for early limb development in arthropods?

Arthropods and in particular crustaceans show a great diversity concerning their limb morphology. This makes the homologization of limbs and their parts and our understanding of evolutionary transformations of these limb types problematical. To address these problems we undertook a comparative study of the limb development of two representatives of branchiopod crustaceans, one with phyllopodous the other with stenopodous trunk limbs. The trunk limb ontogeny of a 'larger branchiopod', Cyclestheria hislopi ('Conchostraca') and the raptorial cladoceran Leptodora kindtii (Haplopoda) has been examined by various methods such as SEM, Hoechst fluorescent stain and expression of the Distal-less gene. The early ontogeny of the trunk limbs in C. hislopi and L. kindtii is similar. In both species the limbs are formed as ventrally placed, elongate, subdivided limb buds. However, in C. hislopi, the portions of the early limb bud end up constituting the endites and the endopod of the phyllopodous filtratory limb in the adult, whereas in L. kindtii, similar limb bud portions end up constituting the actual segments in the segmented, stenopodous, and raptorial trunk limbs of the adults. Hence, the portions of the limbs corresponding to the endites of the phyllopodous trunk limbs in C. hislopi (and other 'larger branchiopods') are homologous to the segments of the stenopodous trunk limbs in L. kindtii. It is most parsimonious to assume that the segmented trunk limbs in L. kindtii have developed from phyllopodous limbs with endites and not vice versa. This study has demonstrated at least one way in which segmented limbs have been derived from phyllopodous, multi-lobate limbs during evolution. Similar pathways can be assumed for the evolution of stenopodous, segmented and uniramous limbs in other crustaceans. Irrespective of the differences in the adult limb morphology, the early patterning of arthropod limbs seems to follow a similar principle.     

Intermolecular plasmid recombination in fibroblasts from humans with DNA damage-processing defects

We have evaluated the ability of immortalized human fibroblasts to recombine transfected plasmid DNA. A number of cell lines from normal individuals and from patients with DNA damage-processing defects were examined. Two plasmid recombination substrates were derived from pSV2neo and contained nonoverlapping deletions in the aminoglycoside phosphotransferase II gene. Intermolecular recombination was assessed by two methods after cotransfection. In a short-term, extrachromosomal recombination assay, low molecular weight DNA was extracted from the human cells 48 h after transfection, and recombinant plasmids were detected by transformation into appropriate indicator bacteria. In a long-term stable recombination assay the fibroblasts were cotransfected and G418-resistant colonies allowed to form. By the former assay all but two cultures were recombination-proficient, whereas all were recombination-proficient by the latter assay. The efficiency of transfection of human cells with plasmids appears to be a major variable affecting recombination. Recombination can be stimulated by uv irradiation of plasmid DNA prior to transfection. Cells from patients with Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum complementation groups A, C, D, E, and G are not defective at intermolecular plasmid recombination.     

Guidelines for the Control of Human Immunodeficiency Virus Infection in Adolescents

Although adolescents account for only 0.4% of reported cases of the acquired immunodeficiency syndrome (AIDS) in the United States, they are sexually active and, therefore, at risk of acquiring human immunodeficiency virus (HIV) infection. To address issues of HIV control in adolescents, we developed guidelines that emphasize education and medical care and deemphasize antibody testing. For adolescents known to be infected with HIV, we recommend no restrictions on access to educational or treatment programs except when their health providers recommend such restrictions to protect them from exposure to opportunistic infections. For adolescents of unknown antibody status with a possible previous exposure to HIV, we recommend that as long as the incidence of HIV infection and clinical AIDS remains low, there should be no restrictions on residential placements and no routine antibody testing.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.     

Alternate pathways of DNA replication in Escherichia coli

We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.     

Characterization of monoclonal antibodies to the IGF-I receptor that inhibit IGF-I binding to human cells

A mouse monoclonal antibody directed against the human placental IGF-I receptor is shown to completely inhibit IGF-I binding to both human placental membranes and circulating human mononuclear leukocytes. Insulin binding is unaffected. Studies with 125I-labelled anti-receptor antibody indicate that the antibody reacts with an epitope distinct from the IGF-I binding site. These antibodies will be powerful probes of IGF-I binding and action in human cells.